Immunocytochemical study of estrogen receptor activation factor (E-RAF) and the proteins that interact with nuclear estrogen receptor II (nER II) in epithelial endometrial cells, in the presence and in the absence of estradiol.

The localization and abundance of the estrogen receptor activation factor (E-RAF) and a small nuclear ribonucleoprotein (snRNP) complex containing three proteins, p32, p55 and p60, which interact with the nuclear estrogen receptor II (nER II), have been studied in rat endometrial epithelial cells by means of immunofluorescence and high resolution quantitative immunocytochemistry. In the cytoplasm E-RAF is associated with the rough endoplasmic reticulum. In the nucleus it is mainly localized at the interchromatin space, and surrounding the clumps of compact or semi-condensed chromatin. Quantitative analyses show that the abundance of E-RAF in the nucleus increases after ovariectomy and decreases 3 minutes after estradiol administration. These results are in agreement with the currently available biochemical data. Double immunolocalizations demonstrate that p32, p55, p60 co-localize with other splicing-related protein. High resolution immunolocalization shows that p32, p55, p60 are associated with perichromatin fibrils (co-transcriptional splicing) and with clusters of interchromatin granules (storage of splicing-related molecules). The nuclear abundance of the snRNP complex decreases with ovariectomy, increases within 3 minutes after estradiol administration and remains higher than that in ovariectomized animals for 27 minutes. These results strongly support the previous data on the role of nER-II in the regulation of mRNA transcription and its export from the nucleus to the cytoplasm.

Proteins interacting with the mammalian estrogen receptor: proposal for an integrated model for estrogen receptor mediated regulation of transcription.

Two forms of estrogen receptor (ER) that exist in the mammalian uterus have been examined in this review. (1) ERalpha, or the classical estrogen receptor that is considered to influence the transcriptional process; (2) the non-activated estrogen receptor (naER), an alternative form of ER with no DNA binding function, localized in the plasma membrane. An integrated model is being proposed to highlight the functional roles of both receptors in transcriptional regulation. The proteins with which ER interacts during various stages of its existence are being examined. These stages include: (1) transport from the cytoplasm to the nucleus; (2) interaction with the nuclear transcription machinery; (3) involvement in post-transcriptional control mechanisms; and (4) degradation through ubiquitination. The proteins with which naER interacts during its plasma membrane-to-nucleus movement have also been identified; the results have not yet been published. Within the nucleus it dimerizes with a DNA-binding protein, the estrogen receptor activation factor (E-RAF). It is being proposed that the purpose behind the dimerization between naER and E-RAF is to transport E-RAF to the transcription initiation site as the naER in the heterodimer is a RNA-polymerase binding protein. Deglycosylated naER fails to dimerize with the E-RAF. Deglycosylation of the naER therefore dissociates the heterodimer and this transformed naER is now identified as nuclear estrogen receptor II (nER II). The dissociated E-RAF is free either to destabilize (E-RAF II) or stabilize (E-RAF I) the DNA while the naER remains bound to the RNA polymerase II. nER II phosphorylates certain subunits in RNA polymerase; the functional significance of this phosphorylation remains to be known.

A nuclear transforming factor that converts the goat uterine nonactivated estrogen receptor to nuclear estrogen receptor II.

A 62-kDa nuclear protein that transforms the goat uterine nonactivated estrogen receptor (naER) to nuclear estrogen receptor II (nER II) has been isolated and purified. This is being identified as the naER-transforming factor (naER-TF). The transformation is achieved through deglycosylation of the naER. It is observed that the naER-TF action on the naER introduces significant changes in the structural and functional features of the naER. The capacity of the naER to bind estradiol increases 8- to 10-fold, while its hormone binding affinity reduces to a considerable extent following its exposure to naER TF. There is a critical ratio in the concentration of the two proteins, the TF and the naER, that would ensure an optimum transformation process. The transformed naER is incapable of dimerization with the estrogen receptor activation factor (E-RAF).

Reverse transcriptase activity in bovine bone marrow: purification of a 66-kDa enzyme.

The presence of reverse transcriptase (RT) activity in a DNA-binding protein complex of the goat bone marrow has been reported earlier from our laboratory. Here we report a procedure for the purification of the enzyme with RT activity from bovine bone marrow and show that the basic function is associated with a approximately 66-kDa protein. This enzyme can use RT specific homopolymers as template and short oligonucleotides as primers, while displaying a Mg(2+)-ion requirement. Eukaryotic RTs have been shown to have endogenous RNAs associated with the enzymes. Evidence is presented here to show that some endogenous RNAs are associated with the RT activity in bovine bone marrow. Even though the enzyme activity appears to be associated with a approximately 66-kDa protein, the results indicate that for a full expression of its activity, the enzyme needs to interact with a 55-kDa protein that co-purifies with the enzyme during ion-exchange chromatography.

Interdependence between a 55 kDa protein (p55) and a 12 kDa protein (p12) in facilitating the nuclear entry of goat uterine estrogen receptor under cell-free conditions.

A 55 kDa nuclear localization signal binding protein (p55) is involved in the transport of the goat uterine estrogen receptor from the cytoplasm to the nuclear pore complex (NPC). p55 forms a complex with a 12 kDa protein (p12) which in turn becomes 'docked' at the NPC. The present study reports on the purification and functional characterization of p12. Both p55 and p12 are Mg2+-dependent ATPases. The protein-protein interactions that take place between these two molecules at the NPC cause an enhancement in the net ATPase activity associated with the protein complex. Presumably, this enhanced ATPase function helps in the final nuclear entry of the estrogen receptor; p55 remains associated with p12 at the nuclear entry site under these conditions.

Cholesterol inhibits the nuclear entry of estrogen receptor activation factor (E-RAF) and its dimerization with the nonactivated estrogen receptor (naER) in goat uterus.

An alternative form of estrogen receptor isolated from goat uterus, the nonactivated estrogen receptor (naER), has no DNA-binding function, although it is closely similar to the classical estrogen receptor (ER) in its hormone binding affinity and specificity. The naER dimerizes with a DNA binding protein, estrogen receptor activation factor (E-RAF). The heterodimer binds to the DNA. Assays carried out during the purification of E-RAF showed that an endogenous inhibitor that is heat stable and dialyzable bound to the E-RAF and prevented the formation of the heterodimer. The inhibitor has been isolated and purified. GC-MS analysis identifies this molecule to be cholesterol. Circular dichroism measurement has shown that the high-affinity binding of cholesterol to E-RAF results in subtle changes in the secondary and the tertiary structure of the protein. The E-RAF with altered conformation fails to dimerize with the naER. Instead of facilitating E-RAF entry into the nucleus, dimerization with the naER prevents it. Similarly, cholesterol binding blocks the nuclear entry of the protein, showing that E-RAF with altered conformation is incapable of interaction with the nuclear pore complex/membrane proteins. The naER-E-RAF heterodimer remains at the nuclear periphery, incapable of further transport. These results indicate the possibility that the dimerization between naER and the E-RAF takes place only within the nuclear compartment. The observation that cholesterol binding prevents nuclear entry of the E-RAF reflects the similarity of E-RAF with the sterol regulatory element (SRE) binding protein that enters the nucleus and binds to SRE only when the intracellular level of cholesterol remains low.

Structural characterization of the goat uterine estrogen receptor activation factor using an endogenous calcium activated neutral protease.

An expedient method for the purification of a calcium activated neutral protease (CANP) of the goat uterus has been designed. This enzyme, purified to homogeneity, has been used as a tool in the structural characterization of the estrogen receptor activation factor II (E-RAF II) that dimerizes with an alternative form of estrogen receptor (ER), the non-activated estrogen receptor (naER). The enzyme cleaves the E-RAF into two fragments, alpha and beta, of molecular mass 32 and 30 kDa, respectively. The beta retains the DNA binding activity, as well as the capacity to dimerize with the naER. On the other hand, the cholesterol binding activity and the ATPase function are shared by both alpha and beta fragments. The E-RAF domain that binds to the nuclear periphery appears to be localized on the beta fragment. The beta fragment, however, is incapable of entering the nucleus on its own.

Plasma membrane is the primary site of localization of the nonactivated estrogen receptor in the goat uterus: hormone binding causes receptor internalization.

Evidence is presented to show that the cellular site of localization of the goat uterine nonactivated estrogen receptor (naER) is the plasma membrane. Exposure of purified plasma membrane preparations to estradiol (20 nM) causes dissociation of the receptor from the membrane into the medium. This receptor movement takes place in the presence of diethylstilbestrol while testosterone, progesterone, dexamethasone, and tamoxifen do not facilitate the dissociation of the naER from the plasma membrane. Further, tamoxifen inhibits the estradiol-mediated dissociation of the naER from the plasma membrane. The naER is a glycoprotein and is also a tyrosine kinase. The tyrosine kinase activity is inhibited in the presence of estrogens while tamoxifen reverses this estrogen-dependent inhibition.

Estrogen receptor activation factor (E-RAF) of the rat uterus: hormonal control.

Evidence is presented showing that the synthesis of the estrogen receptor activation factor (E-RAF) in the rat uterus is under both estrogenic and progestational control. An ELISA method for the measurement of E-RAF is described and used to measure the E-RAF titre in the pregnant rat uterus. It is proposed that E-RAF synthesis during pregnancy is under the exclusive control of progesterone.

Association of cytoskeletal proteins with estrogen receptor in rat uterine cytosol: possible role in receptor movement into the nucleus.

Evidence is presented to demonstrate a close association between an estrogen receptor or an estrogen receptor activation factor and the cytoskeletal proteins, tubulin and actin in rat uterine cytosol. Exposure of uterine cytosol to protein kinase assay medium resulted in the phosphorylation of three proteins, of molecular mass 55, 50 and 45 kDa. These 55 and 50 kDa proteins apparently represent tubulin, while the 45 kDa protein, is presumed to be actin. Phosphorylation of these proteins is under the regulatory influence of estradiol and the cyclic nucleotides, cAMP and cGMP. It is proposed that the protein kinase involved in this process may be the non-activated estrogen receptor (naER).

The nuclear estrogen receptor R-II of the goat uterus: distinct possibility that the R-II is the deglycosylated form of the nonactivated estrogen receptor (naER).

Structural and functional characteristics of the goat uterine nuclear estrogen receptor R-II have been subjected to comparison with those of the nonactivated estrogen receptor (naER), purified from the cytosol. The two proteins have the same molecular mass, 66 kDa; they display identical peptide maps and are both recognized by anti-estrogen receptor (R-I) IgG. Both are tyrosine kinases and bind with equal affinity to a column of anti-phosphotyrosine IgG-Sepharose. On the other hand, while naER is a glycoprotein, the R-II does not show any sign of glycosylation. Unlike the naER, the R-II is incapable of dimerization with estrogen receptor activation factor (E-RAF) and, as a consequence, bind to the DNA. R-II has a higher estradiol binding capacity and the resultant reduction in its affinity for the hormone in comparison with the naER. Further, the sedimentation behavior and the Stokes radius of the naER indicate a globular nature in the shape of the protein. The corresponding data for the R-II reveal that the protein has a distinct nonglobular shape. Deglycosylation of the naER using a glycopeptidase resulted in the total conversion of the distinct physical features of the naER to the R-II category. This treatment resulted, without effecting any reduction in its molecular mass, in the loss of the E-RAF dimerization capacity of the naER. The Stokes radius and the sedimentation coefficient of the protein underwent drastic changes and became closely similar to those of the R-II. In addition, the deglycosylation introduced a several-fold enhancement in the capacity of the naER to bind estradiol with a concomitant decrease in its affinity, similar to the corresponding properties of the R-II. The R-II is shown to have a conformational structure different from that of the naER, to interact with the nuclear RNA polymerase II. It is also shown here that the R-II phosphorylates two subunits (molecular mass 91 and 20 kDa) in the RNA polymerase II, in addition to the 40-kDa subunit phosphorylated by the naER. The results clearly indicate the possibility that the nuclear R-II estrogen receptor is the deglycosylated naER.

Ubiquitination of the rat uterine estrogen receptor: dependence on estradiol.

Using polyclonal antibodies against estrogen receptor and ubiquitin, the ubiquitination of the estrogen receptor has been demonstrated in both in vitro and in vivo conditions. The ubiquitination of the estrogen receptor is estradiol specific and is enhanced by estradiol. Estrogen withdrawal is associated with decreased ubiquitination of the estrogen receptor.

A 55-kDa protein (p55) of the goat uterus mediates nuclear transport of the estrogen receptor. I. Purification and characterization.

A 55-kDa protein (p55), purified from the goat uterine cytosol, transports estrogen receptor (ER) into the nucleus. Selective elution of this protein from a column of estrogen receptor-Sepharose using buffers containing high concentrations of lysine and the high affinity with which it binds to poly-L-lysine-Sepharose indicate that it recognizes a lysine-rich region in the ER. Its strong binding to tubulin-Sepharose and actin-Sepharose is indicative of a role that the cytoskeletal elements play in the nuclear transport of the ER, mediated by p55. This protein can be purified in a single step following chromatography of the uterine cytosol on a column of actin-Sepharose. Antibodies raised against poly-L-aspartic acid cross-reacted with p55 and inhibited the nuclear transport of the ER. The binding of p55 to a heterologous nuclear localization sequence of the SV40 large T suggests that it may also be involved in the transport of proteins other than the ER.

A 55-kDa protein (p55) of the goat uterus mediates nuclear transport of the estrogen receptor. II. Details of the transport mechanism.

A 55-kDa protein (p55) that mediates the transport of the estrogen receptor (ER) from the cytoplasm to the nucleus has been purified to homogeneity from goat uterine cytosol. Using this pure protein, some of the aspects of the mechanisms associated with the transport of the ER to the nucleus have been elucidated. The mechanism of ER transport into the nucleus can be separated into two steps: the p55-mediated transport and binding of ER to the nuclear membrane, followed by an ATP-dependent, 14-kDa protein(s)-mediated translocation of ER into the nucleus. The p55 has inherent ATPase activity and it is proposed that the energy released during this ATP hydrolysis is utilized in the nuclear transport of the ER.

Reverse transcription in a macromolecular complex of ovine bone marrow: possible involvement in the antigen receptor mediated signal transduction process.

A macromolecular fraction was isolated from ovine bone marrow and designated the active fraction (AF). Two distinct enzymatic activities were detected in the AF: (1) a RNA-dependent RNA polymerase, and (2) a reverse transcriptase. RNA polymerase uses an endogenous RNA molecule of the AF as the template while the product of this RNA polymerase reaction forms the template for the reverse transcriptase. Synthetic reactions are initiated exclusively upon the exposure of the in vitro system to one of the external proteins, selected at random, ovalbumin or insulin. Ovalbumin specific (OS) and insulin specific (IS) AF fractions were prepared. OS-AF binds 14C-ovalbumin and the IS-AF binds 14C-insulin, but not vice versa. Reverse transcription activity of OS-AF is stimulated only upon its exposure to ovalbumin and not to insulin while the reverse is true for the activity associated with IS-AF. Indirect evidence indicates that the enzymes which synthesize nucleic acids are closely associated with the antigen receptor on the B-lymphocyte plasma membrane, the mlgM, and that the antigen binding to its receptor helps in the activation of these enzymes.

The nonactivated estrogen receptor (naER) of the goat uterus is a tyrosine kinase.

The nonactivated estrogen receptor (naER) has been isolated and purified to absolute homogeneity from the goat uterine cytosol. It is a 66-kDa protein, sedimenting at 4.2 S on linear sucrose density gradients and having a Stokes radius of 36 A. It displays high affinity and specificity for estradiol and diethyl stilbestrol with a Kd of 1 x 10(-10) M. CNBr peptide analysis reveals that it has a primary structure distinctly different from that of the regular estrogen receptor even though anti-ER antibody cross-reacts with the nonactivated ER. The protein gains access to the DNA only upon dimerization with the estrogen receptor activation factor (E-RAF), a DNA-binding protein having no capacity to bind estradiol. Analysis reveals that both naER and E-RAF are protein kinases. While the E-RAF is a serine kinase, naER functions as a tyrosine kinase. No protein kinase activity is displayed by the regular estrogen receptor. The protein kinase activity of the naER is inhibited in the presence of estradiol. Similarly, the protein kinase activities associated with the proteins disappear when the naER and E-RAF are brought together.

A DNA-binding (R-I) and a non-DNA-binding (R-II) estrogen receptor in the goat uterine nucleus: purification and characterization.

Two forms of nuclear estrogen receptors have been isolated and purified from the goat uterus. The biochemical characteristics of the proteins imply that the receptors may be identified as the type I and type II nuclear estrogen receptors. Nevertheless, we felt a necessity to exercise caution in using this nomenclature and, therefore, decided to identify them instead as R-I and R-II, respectively. While R-I is the DNA-binding form, R-II is a non-DNA-binding protein. The two proteins are totally dissimilar in other physical characteristics like the Stokes radii (36 A for R-I and 21 A for R-II), sedimentation coefficients (4.8 S for R-I and 3.8 S for R-II), the Kd (1 nM for R-I and 2 nM for R-II), and the nature of the CNBr fragmentation of the proteins. The two proteins, however, cross-react with polyclonal antibodies raised against goat uterine estrogen receptor activation factor (E-RAF), a DNA-binding protein with no capacity to bind estradiol, originally discovered by T.N.R.V. Thampan and J. H. Clark (1981, Nature 290, 152-154). A major feature of the R-II isolation procedure is the chromatography of the protein on a heat shock protein 90-Sepharose column in the presence of molybdate ions and elution using a molybdate-free buffer. While estradiol-17 beta (E2) binding to R-II was inhibited by the presence of dithiothreitol and quercetin in the medium, E2-R-I interaction remained unaffected by these exposures.

A four-step, inexpensive protocol for large-scale purification of goat uterine estrogen receptor.

A relatively inexpensive yet highly efficient and extremely rapid procedure has been developed for the isolation and purification of estrogen receptor from the goat uterine cytosol. Greater than 1 mg of purified receptor protein could be obtained from 75 g of uterine tissue within a period of < 24 h, following this protocol. The procedure does not require the use of an ultracentrifuge, a cold room, or a column chromatography setup. The entire isolation procedure, which involves adsorption to and selective elution from different chromatography matrices, is carried out at the laboratory bench using beakers kept in an ice bath. Antibodies raised against this receptor cross-react with the goat uterine estrogen receptor activation factor, a DNA binding protein having no capacity to bind estradiol but which dimerizes with the nonactivated estrogen receptor, an estrogen receptor incapable of binding to DNA on its own.

Synthesis of gap junction proteins and collagenases in the preimplantation rat uterus.

Synthesis of gap junction proteins (GJPs) and of collagenases in the rat uterus has been studied under two physiological conditions: various stages of the estrus cycle, and the early pregnancy period. The synthesis has been studied by incubating uterine horns in a short-term tissue culture medium containing radioactively-labeled amino acids, followed by a double antibody immunoprecipitation of the labeled proteins. After exposure of the media to either anti-collagenase IgG(s) or anti-GJPs IgG(s), the final immunoprecipitation was achieved with the use of goat anti-rabbit IgG. Collagenase(s) synthesis was found to reach the peak, during the estrus cycle, at the proestrus stage, while GJP synthesis reached the maximum during the estrus stage. In the preimplantation, pregnant, rat uterus the syntheses of both the proteins reached the respective peak activities on day 4 of pregnancy, about 24 h before the expected time of ovum implantation. A study of the literature reveals that this time coincides with a spurt in exposure of the progesterone dominated uterus to estradiol.

Hormonal regulation of rat uterine collagenase.

Two different methods of study have been employed in the analysis of the hormonal regulation of rat uterine collagenases. The first is concerned with the enzyme activity and the second, its synthesis. The data presented in this report reveal that the rat uterine collagenase is under the regulatory influence of both estradiol and progesterone. This fact is first exemplified by the observation that on Day 2 post-ovriectomy, a wave of collagenase synthesis takes place which influences the enzyme activity in the uterus during the next 3-4 days. This peak in collagenase synthesis disappeared in the uteri of rats subjected to ovariectomy and adrenalectomy simultaneously. It indicated that a hormone of adrenal origin was responsible for the enhanced synthesis and activity of collagenase in the uteri of ovariectomized rats. That the hormone involved was progesterone was shown by the subsequent experimental data. The estradiol-mediated enhancement in rat uterine collagenase activity has been shown to be inhibited by intraluminal exposure of the uteri to actinomycin D/cyclohexinmide, indicating the apparent influence of the hormone at the level of the collagenase gene.